Macrocyclic difluorostatone derivatives useful as antiviral agents

ABSTRACT

The present invention provides novel macrocyclic difluorostatone derivatives which are useful as antiviral agents. More specifically, these novel compounds are useful as inhibitors of retroviral proteases required for replication, particularly the HIV-1 and HIV-2 viral proteases, in the prevention or treatment of infection by the human immunodeficiency virus (HIV), and in the treatment of consequent pathological conditions such as the acquired immunodeficiency syndrome (AIDS) in mammals capable of being infected with HIV virus.

This application is a continuation of application Ser. No. 08/192,080,filed Feb. 4, 1994, now abandoned, and had an effective internationalfiling date of Nov. 28, 1994, as application PCT/US94/13711, whichdesignated the U.S. and entered the U.S. national phase on Sep. 9, 1996under 35 USC 371.

BACKGROUND OF THE INVENTION

A great deal of research is currently underway to develop treatments andcures for viral infections in humans and in animals. Notably theincidence of AIDS and ARC in humans is increasing at an alarming rate.The five year survival rate for those with AIDS is dispiriting and AIDSpatients, whose immune systems have been seriously impaired by theinfection, suffer from numerous opportunistic infections includingKaposi's sarcoma and Pneumocystis carninii pneumonia. No cure for AIDSis known and current treatments are largely without adequate proof ofefficacy and have numerous untoward side effects. Fear of the diseasehas resulted in social ostracism of and discrimination against thosehaving or suspected of having the disease.

The present invention relates to compounds that are useful as antiviralagents. More specifically this invention relates to macrocyclicdifluorostatone derivatives that are useful as inhibitors of retroviralproteases required for replication, such as the HIV-1 and HIV-2 viralproteases, the prevention or treatment of infection by the humanimmunodeficiency virus (HIV), and the treatment of consequentpathological conditions such as the acquired immunodeficiency syndrome(AIDS) in mammals capable of being infected with HIV virus.

SUMMARY OF THE INVENTION

The present invention relates to compounds having the following generalformula I: ##STR1## and the stereoisomers, hydrates, andpharmaceutically acceptable salts thereof wherein

P₂ is C₁₋₆ alkyl, cyclopentyl, hydroxy C₁₋₆ alkyl, phenyl, benzyl or3-tetrahydrofuryl;

P₃ is selected from the group consisting of hydrogen, --CH₃, --CH(CH₃)₂,--CH₂ CH(CH₃)₂, --CH(CH₃)(CH₂ CH₃), --CH₂ SH, --CH₂ CH₂ SCH₃, --CH₂ OH,--CH(CH₃)OH, --CH₂ (CH₂)₃ NH₂, --CH₂ (CH₂)₂ NHC (═NH) NH₂, --CH₂ CO₂ H,--CH₂ CH₂ CO₂ H, --CH₂ CONH₂, --CH₂ CH₂ CONH₂, benzyl, ##STR2## R₁ ishydrogen, C₁₋₁₅ alkyl, hydroxy C₁₋₁₅ alkyl, CH([(CH₂)_(d) -O--CH₂ ]_(f)-R₇)₂, CH₂ Si(CH₃)₂ (R₈), PDL, --(C₁₋₆ alkylene)--OR₄, CH(Y)(Z),##STR3## wherein PDL is --(CH₂)_(a) -2-, 3- or 4-pyridyl, Y is hydroxyC₁₋₁₅ alkyl, C₁₋₆ alkyl or (CH₂)_(e) -C₆ H₄ -(V)_(e') ; Z is (CH₂)_(d)-O--CHO, C₁₋₆ alkylene-O-(CH₂)_(d) -(O--CH₂ --CH₂)_(e) -O--C₁₋₆ alkyl,CHO, CO₂ R₄, CONHR₄, (CH₂)_(d) -O--(CH₂)_(d') -R₅, (CH₂)_(e) -OR₄ or##STR4## wherein V is OR₄ or hydroxy C₁₋₆ alkylene; provided that d'=2when R₅ is piperazinyl, substituted piperazinyl, piperidyl ormorpholinyl;

R₂ is as defined for R₁ with the proviso that R₂ is other than hydrogenwhen R₁ is hydrogen, or R₁ and R₂ taken together with the nitrogen atomto which they are attached are selected from the group consisting of;##STR5## R₃ is CH₂ OR₄, C(O)NHR₄ or CHO; R₄ is hydrogen, C₁₋₆ alkyl,phenyl or benzyl;

R₅ is piperazinyl, substituted piperazinyl, piperidyl, morpholinyl,pyridyl, pyrazinyl, pyrimidinyl or phenyl, wherein substitutedpiperazinyl is piperazinyl substituted on one nitrogen atom thereof withCHO, C(O)NHR₄, C₁₋₄ alkyl or CO₂ R₄ ;

R₆ is (H, OH) or ═O;

R₇ is pyrimidyl, pyridyl, pyrazinyl or phenyl;

R₈ is C₁₋₆ allenyl, C₁₋₆ alkoxy, C₁₋₆ alkylene, hydroxy C₁₋₆ alkyl, C₁₋₆alkyl, or OH;

a is zero, 1, 2 or 3;

b is zero or 1;

d and d' are each independently 1 or 2;

e and e' are each independently zero, 1 or 2;

f is zero or one; and

x is 1, 2, 3, or 4.

The present invention further provides a method of treating a patientsuffering from a viral infection comprising administering to saidpatient an effective antiviral amount of a compound of formula (I).

In addition the present invention provides a method of inhibiting HIVprotease in a patient in need thereof comprising administering to saidpatient an effective inhibitory amount of a compound of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

The term "halogen", "halo" or "halide" refers to a chlorine, bromine oriodine atom. The term "stereoisomer" refers to a compound made up of thesame atoms bonded by the same bonds but having differentthree-dimensional structures which are not interchangeable. The threedimensional structures are called configurations. The term"diastereomer" refers to those stereoisomers with more than one chiralcenter that are not mirror images of one another. The term "enantiomer"refers to two stereoisomers whose molecules are nonsuperimposable mirrorimages of one another. The term "racemic mixture" or "racemicmodification" refers to a mixture of equal parts of enantiomers. Theterm "chiral center" refers to a carbon atom to which four differentgroups are attached. For amino acids, the designations L/D or R/S can beused as described in IUPAC-IUB Joint Commission on BiochemicalNomenclature, Eur. J. Biochem., 138, 9-37 (1984). It is understood thatthe compounds of formula (I) may exist in a variety of stereoisomericconfigurations. It is further understood that where the configuration offormula (1) is fixed, the maximum number of enantiomers possible foreach compound is equal to 2^(n) wherein n represents the total number ofchiral centers located on the compound. The minimum number of chiralcenters located on formula (I) are indicated below by the * ##STR6##wherein the substituents are previously defined provided P₃ is otherthan hydrogen.

A compound of the invention may be in free form, e.g., amphoteric form,or in salt, e.g., acid addition or anionic salt, form. A compound infree form may be converted into a salt form in an art-known manner andvice-versa.

The pharmaceutically acceptable salts of the compounds of formula I (inthe form of water, or oil-soluble or dispersible products) include theconventional non-toxic salts or the quaternary ammonium salts of thesecompounds, which are formed, e.g., from inorganic or organic acids orbases. Examples of such acid addition salts include acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, paemoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, the basic nitrogen-containinggroups may be quaternized with such agents as lower alkyl halides, suchas methyl, ethyl, propyl, and butyl chloride, bromides and iodides;dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates,long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides and others.

The hydrates of the compounds of formula (I) are hydrated ketonescompounds having the partial structure ##STR7## and in their end-useapplication are generally the active forms.

In general, as used herein, the term "alkyl" includes the straight,branched-chain and cyclized manifestations thereof unless otherwiseindicated, particularly such moieties as methyl, ethyl, isopropyl,n-butyl, t-butyl, --CH₂ -t-butyl, cyclopropyl, n-propyl, pentyl,cyclopentyl, n-hexyl, cyclohexyl and cyclohexylmethyl. The term"aralkyl", when used, includes those aryl moieties attached to analkylene bridging moiety, preferably methylene or ethylene.

"Aryl" includes both carbocyclic and heterocyclic moieties of whichphenyl, pyridyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, furyl andthienyl are of primary interest; these moieties being inclusive of theirposition isomers such as, for example, 2-, 3-, or 4-pyridyl, 2- or3-furyl and thienyl, 1-, 2-, or 3-indolyl or the 1- and 3-indazolyl, aswell as the dihydro and tetrahydro analogs of the furyl and thienylmoieties. Also included within the term "aryl" are such fusedcarbocyclic moieties as pentalenyl, indenyl, naphthalenyl, azulenyl,heptalenyl, acenaphthylenyl, fluorenyl, phenalenyl, phenanthrenyl,anthracenyl, acephenanthrylenyl, aceanthrylenyl, triphenylenyl, pyrenyl,chrysenyl and naphthacenyl. Also included within the term "aryl" aresuch other heterocyclic radicals as 2- or 3-benzo[b]thienyl, 2- or3-naphtho[2,3-b]thienyl, 2- or 3-thianthrenyl, 2H-pyran-3-(or 4- or5-)yl, 1-isobenzo-furanyl, 2H-chromenyl-3-yl, 2- or 3-phenoxathiinyl, 2-or 3-pyrrolyl, 4- or 3-pyrazolyl, 2-pyrazinyl, 2-pyrimidinyl,3-pyridazinyl, 2-indolizinyl, 1-isoindolyl, 4H-quinolizin-2-yl,3-isoquinolyl, 2-quinolyl, 1-phthalazinyl, 1,8-naphthyridinyl,2-quinoxalinyl, 2-quinazolinyl, 3-cinnolinyl, 2-pteridinyl,4aH-carbazol-2-yl, 2-carbazolyl, β-carbolin-3-yl, 3-phenanthridinyl,2-acridinyl, 2-perimidinyl, 1-phenazinyl, 3-isothiazolyl,2-phenothiazinyl, 3-isoxazolyl, 2-phenoxazinyl, 3-isochromanyl,7-chromanyl, 2-pyrrolin-3-yl, 2-imidazolidinyl, 2-imidazolin-4-yl,2-pyrazolidinyl, 3-pyrazolin-3-yl, 2-piperidyl, 2-piperazinyl,1-indolinyl, 1-isoindolinyl, 3-morpholinyl, benzo[b]isoquinolinyl andbenzo[b]furanyl, including the position isomers thereof except that theheterocyclic moieties cannot be attached directly through their nitrogenone, two or three substituents independently selected from C₁₋₆ alkyl,haloalkyl, alkoxy, thioalkoxy, aminoalkylamino, dialkylamino, hydroxy,halo, mercapto, nitro, carboxaldehyde, carboxy, carboalkoxy andcarboxamide.

Likewise the term "alkylene" includes straight or branched-chainmoieties. Some examples of branched-chain alkylene moieties areethylethylene, 2-methyltrimethylene, 2,2-dimethyltrimethylene, and soon. For example, C₃ alkylene can mean ##STR8##

All (C₁₋₁₅) moieties are preferably (C₁₋₆) moieties and all (C₁₋₆)moieties such as C₁₋₆ alkyl, C₁₋₆ allenyl, C₁₋₆ alkoxy, and hydroxy C₁₋₆alkyl, are more preferably C₁₋₃ moieties (containing 1-3 carbon atomsinstead of 1-6 carbon atoms).

The fluorenylmethyloxy moiety is that moiety generally called by itsabbreviation FMOC, and is the fluorenyl moiety bearing --CH₂ O attachedto the 9-position of the fluoroenyl moiety. Other terms defined hereinare piperazinyl ##STR9## or substituted piperazinyl ##STR10## thesubstitution (★) occuring only at one nitrogen molecule which is notattached to the remainder of the molecule (attachment via a nitrogenatom). The substituents are one of CHO, C(O)NHR₄, C₁₋₄ alkyl or CO₂ R₄.

More specifically, in the instance wherein P₂ is either C₁₋₆ alkyl orhydroxy C₁₋₆ alkyl, such moieties as --C(CH₃)₃, --CH(CH₃)₂, --CH(CH₃)(C₂H₅), --C(OH)(CH₃)₂ and --CH(OH)CH₃ are preferred.

Piperidyl and morpholinyl both bind to the rest of the ##STR11##molecule via their respective nitrogen atoms while pyrimidinyl, pyridyland pyrazinyl bind to the rest ##STR12## of the molecule anywhere excepttheir respective nitrogen atoms. and the hydroxy radical is not limitedto the terminal carbon atom of the alkyl moiety).

As used herein the term "Pg" refers to a protecting group. Among theclasses of amino protecting groups contemplated are: (1) acyl typeprotecting groups such as formyl, trifluoroacetyl, phthalyl,p-toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfenyl,tritylsulfenyl, and O-nitrophenoxyacetyl; (2) aromatic urethane typeprotecting groups such as benzyloxycarbonyl and substitutedbenzyloxycarbonyls such as p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α-,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, and benzhydryloxycarbonyl;(3) aliphatic urethane protecting groups such as tert-butyloxycarbonyl(Boc), 9-fluorenylmethoxycarbonyl (FMOC), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, and allyloxycarbonyl; (4)cycloalkyl urethane type protecting groups such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;(5) thio urethane type protecting groups such as phenylthiocarbonyl; (6)alkyl type protecting groups such as triphenylmethyl (trityl) and benzyl(Bzl); (7) trialkylsilane protecting groups such as trimethylsilane ifcompatible. The preferred α-amino protecting groups aretert-butyloxycarbonyl (Boc) or benzyloxycarbonyl (CBZ). The use of Bocas an α-amino protecting group for amino acids is described by Bodanskyet al. in "The Practice of Peptide Synthesis", Springer-Verlag, Berlin(1984), p. 20.

Where functional groups other than the α-amino group are present, suchas those that may be present on P₃, those groups will generally have tobe protected. These functional groups may be protected by differentprotecting groups from those used on the a-amino groups so that oneprotecting group can be removed without removing the other protectinggroup. The selection of appropriate combinations of protective groupsand reagents to selectively remove protective groups is well known inthe art. For example, see M. Bodansky, "Peptide Chemistry, A PracticalTextbook", Springer-Verlag (1988); J. Stewart, et al., "Solid PhasePeptide Synthesis", 2nd ed., Pierce Chemical Co. (1984).

In general the compounds of this invention may be prepared usingstandard chemical reactions analogously known in the art. Morespecifically, the preparation of compounds of structure (3) is wellknown in the art and described generally by Schirlin, D. and VanDorsselaer, V. in PCT/US91/09741 published Jul. 23, 1992 with aninternational publication number of WO 92/12123. For example, thecompounds of structure (3) and (4) which are required starting materialfor use in Scheme II, can be prepared as described in Scheme I. The term"Pg'" as used in Schemes I and II is a protecting group as previouslydefined but does not include benzyl or the aromatic urethane protectinggroups described. All other substituents, unless otherwise indicated,are previously defined. The reagents and starting materials are readilyavailable to one of ordinary skill in the art. ##STR13##

In Scheme I step A, the aldehyde (1) is subjected to a condensationreaction with an ester of bromodifluoroacetic acid, preferably the ethylester in the presence of zinc and in an anhydrous aprotic solvent, suchas tetrahydrofuran, diethyl ether, t-butyl methyl ether and the likeunder a nitrogen or argon inert atmosphere. The reaction is gentlyheated to about 60° C. for about 1-12 hours or ultrasonicated to producethe ester described by (2). The preferred amino protecting group (Pg')on the aldehyde (1) is the tert-butyloxycarbonyl group.

Alternatively, in Scheme I step A, the condensation to produce ester (2)can be achieved in greater yields and at lower reaction temperaturesutilizing the following general method. Under an inert atmosphere, suchas nitrogen, the aldehyde (1) is dissolved in a suitable anhydrousorganic solvent. Examples of a suitable anhydrous organic solvent aretetrahydrofuran, diethyl ether, t-butyl methyl ether and the like. Thesolution is cooled to approximately 0° C. To the solution is added about0.30 equivalents of silver acetate, about 2.1 equivalents of zinc dust,and about 2 equivalents of ethyl bromodifluoroacetate. About 0.34equivalents of diethylaluminum chloride (as a solution in toluene) isadded slowly to the reaction keeping the temperature of the reactionbelow 12° C. The reaction is allowed to stir for 1 to 3 hours at about0° C. and then at room temperature for 4 to 12 hours. The reaction isthen cooled to about 10° C. and quenched with saturated aqueous ammoniumchloride. The ester (2) is then isolated and purified by techniques wellknown in the art. For example a solution of sodium hydrogen tartrate isadded and the reaction is allowed to warm from 10° C. to roomtemperature. The mixture is filtered, the solids washed with a suitableorganic solvent, such as ethyl acetate and the layers of the filtrateare separated. The aqueous layer is extracted with ethyl acetate, theorganic layer and extracts are combined, dried over anhydrous magnesiumsulfate, filtered and concentrated. The residue if purified by flashchromatography on silica gel with a suitable eluent, such ascyclohexane/ethyl acetate to provide the ester (2).

In Scheme I step B the ester (2) is subjected to an amidation reactionto provide the amide described by structure (3). The ester (2) isdissolved in a suitable organic solvent, such as tetrahydrofuran andtreated with the appropriate R₁,R₂ -substituted amine at a temperatureof from 0 to 80° C. to provide the amide (3).

Alternatively, an appropriate R₁,R₂ -substituted amine that is protectedas necessary is dissolved in a suitable organic solvent, such asdichloromethane under an inert atmosphere, such as nitrogen. Anequivalent of a 2M solution of trimethylaluminum in toluene is addeddropwise to the solution. After approximately 15 minutes this solutionis added to approximately 0.3 equivalents of ester (2) dissolved in asuitable organic solvent, such as dichloromethane. The reaction isallowed to stir for about 15 to 24 hours at about room temperature to40° C. The product is then isolated using techniques well known in theart. For example cold dilute aqueous hydrochloric acid and ethyl acetateis added. The organic layer is separated and washed with water, brine,dried over anhydrous magnesium sulfate, filtered and concentrated undervacuum to provide the amide (3).

Alternatively, the ester (2) may be hydrolyzed to the corresponding acidunder conditions well known in the art and subsequently coupled to theappropriate R₁,R₂ -substituted amine utilizing peptide forming couplingprocedures that are well known in the art to provide the amide (3).

In Scheme I step C, the phenolic ether portion of the ester (2) isdebenzylated under conditions well known in the art to provide thephenol described by structure (2a). For example, the ester (2) isdissolved in a suitable solvent mixture, such as 4.4% formicacid/methanol. A catalytic amount of palladium black is added inportions during a period of about 1 hour to 6 days until debenzylationis complete as indicated by thin layer chromatography or HPLC. Theproduct is then isolated and purified by techniques well known in theart such as flash chromatography. For example, the reaction is filtered,the filtrate concentrated under vacuum and the residue purified by flashchromatography on silica gel utilizing a suitable eluent, such ascyclohexane/ethyl acetate to provide the phenol (2a).

In Scheme I step D, the phenol (2a) is subjected to an amidationreaction to provide the amide described by structure (4). For example,an appropriate R₁,R₂ -substituted amine that is protected as necessary,such as O-benzyl-D-valinol is dissolved in a suitable organic solvent,such as dichloromethane under an inert atmosphere, such as nitrogen. Anequivalent of a 2M solution of trimethylaluminum in toluene is addeddropwise to the solution. After approximately 15 minutes this solutionis added to approximately 0.3 equivalents of (2a) dissolved in asuitable organic solvent, such as dichloromethane. The reaction isallowed to stir for about 15 to 24 hours at about room temperature to40° C. The product is then isolated using techniques well known in theart. For example cold dilute aqueous hydrochloric acid and ethyl acetateis added. The organic layer is separated and washed with water, brine,dried over anhydrous magnesium sulfate, filtered and concentrated undervacuum to provide the amide (4).

The compounds of formula (I) can be prepared as described in Scheme II.All substituents, unless otherwise indicated, are previously defined.The reagents and starting materials are readily available to one ofordinary skill in the art. ##STR14##

In Scheme II step A, the amide (3) is debenzylated to provide the phenoldescribed by structure (4). For example, following generally theprocedure of El Amin et al. J. Org. Chem., 44, 3442 (1979), the amide(3) is dissolved in a suitable solvent mixture, such as 4.4% formicacid/methanol to which a catalytic amount of Pd black has been added.The reaction is stirred for about 4 to 6 hours, with additional portionsof Pd black being added as needed, at intervals of about every 45minutes until the reaction is complete. The reaction is then filteredand the filtrate is concentrated under vacuum. The residue is purifiedby techniques well known in the art, such as recrystallization. Forexample, the residue is recrystallized from a suitable solvent mixture,such as cyclohexane/ethyl acetate, to provide phenol (4).

In Scheme II step B, the phenol (4) is alkylated to provide the etherdescribed by structure (5). For example, the phenol (4) is dissolved ina suitable organic solvent, such as acetone. Approximately 1.2equivalents of a suitable base, such as potassium carbonate, are addedfollowed by addition of approximately 1.15 equivalents of a suitablealkyl halide. Examples of suitable alkyl halides are ethyl bromoacetate,methyl bromoacetate, ethyl 3-bromopropionate, ethyl 3-chloropropionate,ethyl 4-bromobutyrate, ethyl 4-chlorobutyrate, ethyl 5-bromovalerate andthe like. A catalytic amount of potassium iodide is then added and thereaction is stirred for 1 to 3 days. The product is isolated andpurified by techniques well known in the art, such as extractive methodsand recrystallization. For example, the reaction is poured into asuitable solvent mixture, such as ethyl acetate/dilute aqueous sodiumchloride and the organic layer is separated. The organic layer is thenwashed with dilute aqueous potassium hydroxide, brine, dried overanhydrous magnesium sulfate, filtered and concentrated under vacuum. Theresidue is purified by recrystallization from a suitable solventmixture, such as cyclohexane/ethyl acetate to provide the ether (5).

In Scheme II step C, the protected amine portion of ether (5) isdeprotected under conditions well known in the art as described by T. H.Green, "Protective Groups in Organic Synthesis", John Wiley and Sons,1981, Chapter 7, to provide the deprotected amine described by structure(6). For example when Pg' is t-butyloxycarbonyl, the ether (5) istreated with excess trifluoroacetic acid (TFA) and the reaction isallowed to stir for approximately 2 hours under an atmosphere ofnitrogen. The reaction is then concentrated under vacuum. The residue istwice dissolved in ethyl acetate and each time concentrated under vacuumto provide the deprotected amine (6) as the TFA salt. Alternatively whenPg' is t-butyloxycarbonyl, the ether (5) may be treated with excessformic acid and allowed to stir for about 1 to 2 hours at roomtemperature. The deprotected amine (6) can be isolated by treatment withaqueous sodium bicarbonate and extraction with a suitable organicsolvent, such as ethyl acetate. The organic extract is dried overanhydrous magnesium sulfate, filtered and concentrated under vacuum toprovide the deprotected amine (6).

In Scheme II step D, the deprotected amine (6) is immediately subjectedto a coupling reaction [to avoid possible lactamization of (6)] with anacid of structure (6a) ##STR15## under conditions well known in the artto provide the amide described by structure (7) wherein P₃ isappropriately protected as required to prevent formation of undesiredbonds.

P₃ requires an appropriate protecting group when P₃ is --CH₂ SH, --CH₂OH, --CH(CH₃)OH, --CH₂ (CH₂)₃ NH₂, --CH₂ (CH₂)₂ NHC(═NH)NH₂, --CH₂ CO₂H, --CH₂ CH₂ CO₂ H, ##STR16## otherwise P₃ is not protected. Theprotecting groups that can be used, their selection and subsequentremoval is well within the scope of the art, for example see T. H.Greene, "Protective Groups in Organic Chemistry", John Wiley & Sons, NewYork (1981); "The Peptides: Analysis, Synthesis, Biology", Vol. 3,Academic Press, New York (1981); M. Bodansky, "Peptide Chemistry, APractical Textbook", Springer-Verlag (1988); and J. Stewart, et al.,"Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co. (1984).

The selection of the appropriate coupling reaction procedure is withinthe skill of the art. The coupling reaction can be carried out usingstandard coupling procedures such as the azide method, mixed carbonicacid anhydride (isobutyl chloroformate) method, carbodiimide(dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-solublecarbodiimide) method, active ester (p-nitrophenyl ester,N-hydroxy-succinic imido ester) method, Woodward reagent K method,carbonyldiimidazole method, phosphorus reagents such as BOP-Cl, oroxidation-reduction methods. Some of these methods (especially thecarbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole.For example, the deprotected amine (6) [as the free base or the TFAsalt] is dissolved in a suitable organic solvent mixture, such asmethylene chloride/dimethylformamide (1:1) with stirring under an inertatmosphere, such as nitrogen. Approximately 1.06 equivalents of1-hydroxybenzotriazole hydrate (HOBT) are added followed by addition ofN-methylmorpholine [1.1 equivalents if (6) is a free base and 2.2equivalents if (6) is the TFA salt], approximately 1.06 equivalents of(6a) and approximately 1.11 equivalents of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). Thereaction is allowed to stir for about 12 hours to 3 days. The product isthen isolated and purified by techniques well known in the art such asextractive methods, flash chromatography and recrystallization. Forexample, the reaction is poured into water and the mixture is extractedwith a suitable organic solvent, such as ethyl acetate. The organicextract is washed with dilute aqueous hydrochloric acid, aqueous sodiumbicarbonate, brine, dried over anhydrous magnesium sulfate, filtered andconcentrated under vacuum. The residue is then purified by flashchromatography utilizing a suitable eluent, such as ethylacetate/cyclohexane on a stationary phase of silica gel followed bycrystallization from a suitable solvent mixture, such as ethylacetate/cyclohexane to provide the amide (7).

In Scheme II steps E and F the ester portion of amide (7) is convertedto the activated pentafluorophenyl ester described by structure (8). Forexample, the amide (7) is suspended in a suitable solvent mixture, suchas methanol/water (19:1). Approximately 1.4 equivalents of a suitablebase, such as lithium hydroxide are added with stirring. The reaction isallowed to stir for about 2 to 4 hours. The reaction is thenconcentrated under vacuum. The resulting salt of the corresponding acidis purified by techniques well known in the art. For example, the saltis dissolved in water and washed with ether. A suitable organic solvent,such as ethyl acetate is then added to the aqueous phase and 0.1N sodiumbisulfate is added with vigorous stirring until the aqueous phase becomeacidic. The organic layer is then separated, dried over anhydrousmagnesium sulfate, filtered and concentrated under vacuum to provide thecorresponding acid. The acid is then dissolved in methylene chloride. Tothis solution is added approximately 1.3 equivalents ofpentafluorophenol and approximately 1.2 equivalents of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride withstirring. The reaction is allowed to stir for about 3 hours to 3 days.The product is then isolated and purified by techniques well known inthe art. For example, the reaction is diluted with water and theresulting solid is then collected by filtration followed by rinsing withwater and ether. It can then be recrystallized from a suitable solventmixture, such as cyclohexane/ethyl acetate to provide thepentafluorophenyl ester (8).

In Scheme II step G, the protected amine portion of thepentafluorophenyl ester (8) is deprotected under conditions well knownin the art as described by T. H. Green, "Protective Groups in OrganicSynthesis", John Wiley and Sons, 1981, Chapter 7, to provide thedeprotected amine described by structure (9). For example when Pg ist-butyloxycarbonyl, the pentafluorophenyl ester (8) is treated withexcess 4N hydrogen chloride/dioxane with stirring. The reaction isallowed to stir for 30 minutes to 2 hours. The reaction is thenconcentrated under vacuum to provide the deprotected amine (9) as thehydrochloride salt.

In Scheme II step H, the deprotected amine (9) hydrochloride salt issubjected to a cyclization reaction to provide the macrocyclic alcoholdescribed by structure (10). For example, the deprotected amine (9) istreated with a suitable base and organic solvent mixture, such as diluteaqueous sodium bicarbonate/methylene chloride. The reaction is stirredvigorously for 1 to 3 days. The product is then isolated and purified bytechniques well known in the art. For example, the reaction is thenfiltered and the solid is rinsed with water and ether, to provide themacrocyclic alcohol (10) which can be purified by techniques well knownin the art.

An alternative method for converting the amide (7) to the macrocyclicalcohol (10) can be accomplished in two steps. When Pg is an FMOCprotecting group on amide (7), treatment of amide (7) with approximately2 equivalents of a suitable base, such as lithium hydroxide will providethe acid and deprotected amine of structure (7a). ##STR17## Subjecting(7a) to standard coupling conditions as previously described in SchemeII, step D results in cyclization of (7a) to provide the macrocyclicalcohol (10).

In Scheme II step I, the macrocyclic alcohol (10) is oxidized underconditions well known in the art to provide the macrocyclic ketone offormula (Ia) when P₃ is not protected or the macrocyclic ketone ofstructure (11) when P₃ is appropriately protected. For example, themacrocyclic alcohol (10) is dissolved in a suitable organic solventmixture, such as dimethyl sulfoxide/methylene chloride (3:1) under anatmosphere of nitrogen and cooled to approximately -15 to -17° C.Approximately 9 equivalents of oxalyl chloride are added dropwise to thesolution. After about 1 hour approximately 19 equivalents oftriethylamine are added to the reaction which is then allowed to slowlywarm to room temperature and stir for about 17 hours. The product isthen isolated and purified by techniques well known in the art, such asextractive methods, flash chromatography and recrystallization. Forexample, the reaction is diluted with a suitable solvent mixture, suchas water/ethyl acetate. The organic layer is separated and washed withwater, brine, dried over anhydrous magnesium sulfate, filtered andconcentrated under vacuum. The residue is purified by flashchromatography utilizing a suitable eluent, such as ethylacetate/methanol (19:1) and subsequent recrystallization from a suitablesolvent mixture, such as ethyl acetate/2,2,2-trifluoroethanol to providethe macrocyclic ketone of formula (Ia) or macrocyclic ketone (11).

Alternatively the oxidation can be carried out with the Dess-Martinperiodinane (i.e.,1,1,1-triacetoxy-1,1-dihydro-2,1-benzoxiodol-3(1H)-one), [see DessMartin, J. Org. Chem., 48, 4155, (1983)]. This oxidation is effected bycontacting about 1 equivalent of the alcohol with 1 to 10 equivalents ofperiodinane (preferably greater than 5 equivalents), said reagent beingin suspension in an inert solvent (e.g., methylene chloride) under aninert atmosphere (preferably nitrogen) under anhydrous conditions at 0°C. to 50° C. (preferably room temperature) and allowing the reactants tointeract for about 1 to 48 hours. The desired ketone can then beisolated and purified by techniques well known in the art as describedabove.

In Scheme II step J, the protected portion of P₃ on the macrocyclicketone (11) is deprotected under conditions well known in the art toprovide the macrocyclic ketone of formula (Ib).

In Scheme III an alternative method for the preparation of compounds offormula (I) is described wherein the deprotected amine (6) prepared inScheme II is the starting material. All other substituents, unlessotherwise indicated, are previously defined. The reagents and startingmaterials are readily available to one of ordinary skill in the art.##STR18##

In Scheme III step A, the deprotected amine is subjected to a couplingreaction with an acid of structure (6b) ##STR19## under the couplingconditions described previously in Scheme II step D to provide the amideof structure (12).

In Scheme III step B the amide (12) is deprotected under the conditionsdescribed in Scheme II step C to provide the deprotected amine, which issubsequently subjected to a coupling reaction with an acid of structure(6c) ##STR20## under the coupling conditions described previously inScheme II step D to provide the amide of structure (7). The amide (7) isthen converted to compounds of formula (I) as previously described inScheme II.

The diastereomers of formula (I) can be separated and the enantiomers offormula (I) can be resolved utilizing techniques well known in the artsuch as the crystallization techniques described by Jacques, J. et al."Enantiomers, Racemates, and Resolutions", John Wiley and Sons, Inc.,1981 or by chromatography utilizing a suitable stationary phase, such asa chiral stationary phase under HPLC (high pressure liquidchromatography) conditions or flash chromatography.

The following examples present typical syntheses as described by SchemesI, II and III. These examples are understood to be illustrative only andare not intended to limit the scope of the invention in any way. As usedin the following examples, the following terms have the meaningsindicated: "eq." refers to equivalents, "g" refers to grams, "mg" refersto milligrams, "mmol" refers to millimoles, "mL" refers to milliliters,"°C." refers to degrees Celsius, "TLC" refers to thin layerchromatography, "δ" refers to parts per million down field fromtetramethylsilane for ¹ H NMR and "δ" refers to parts per millionupfield from fluorotrichloromethane for ¹⁹ F NMR.

EXAMPLE 1 Preparation of[9(S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-(phenylmethyl)-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide##STR21## Preparation of the starting material in Scheme I,O-benzyl-N-(tert-butoxycarbonyl)-L-tyrosinal (1) [Following theprocedure of Schirlin, D. and Van Dorsselaer, V. in PCT/US91/09741published Jul. 23, 1992 with an international publication number of WO92/12123.]

A mixture of N-tert-butoxycarbonyl-L-O-benzyltyrosine (37.1 g, 100mmol), dicyclohexylcarbodiimide (20.6 g, 100 mmol), andN-hydroxybenzotriazole hydrate (15.3 g, 100 mmol) in anhydrousdichloromethane (350 mL) is stirred at 0° C. for 10 minutes. To this isadded at 0° C., N,O-dimethylhydroxylamine hydrochloride (9.75 g, 100mmol) and N-methylmorpholine (10.1 g, 100 mmol). The temperature isallowed to warm to room temperature and stirring is continued for 15hours. The white precipitate is then filtered off and rinsed withdichloromethane. The filtrate is concentrated under vacuum and theresidue is purified by flash chromatography (silica gel, ethylacetate/cyclohexane, 2:8) to provide theN-tert-butoxycarbonyl-L-O-benzyltyrosine-N,O-dimethyl-hydroxamate (34.3g) as a white solid (R_(F) =0.36 in ethyl acetate/cyclohexane, 1:1).

The N-tert-butoxycarbonyl-L-O-benzyltyrosine-N,O-dimethyl-hydroxamate(18.2 g, 44 mmol) is dissolved in a mixture of anhydrous diethylether/dimethoxyethane (300 mL, 4:1) and cooled to 0° C. To this is addedlithium aluminum hydride (1.82 g, 48 mmol) portionwise. The reaction isstirred at 0° C. for 1.5 hours. A 1M solution of potassium hydrogensulfate (55 mL) is then added dropwise with stirring to the reaction.After addition is complete, the aqueous phase is decanted and extractedwith ethyl acetate (2×200 mL). The combined organic layers are washedwith 3N hydrochloric acid (250 mL), water (200 mL), saturated sodiumbicarbonate (150 mL) and brine (200 mL). The organic layer is then driedover anhydrous magnesium sulfate, filtered and concentrated undervacuum. The residue is recrystallized from ethyl acetate/pentane toprovide N-tert-butoxycarbonyl-L-O-benzyltyrosinal (13 g).

Preparation of4-tert-butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenylpentanoicacid, ethyl ester

Scheme I step A; To a stirred mixture ofN-tert-butoxycarbonyl-L-O-benzyltyrosinal (13.0 g, 36.6 mmol), silveracetate (1.82 g, 10.9 mmol), activated zinc dust (5.02 g, 76.8 mg-atom,washed with 3N hydrochloric acid, water, acetone and ether) and ethylbromodifluoroacetate (14.8 g, 72.9 mmol) in anhydrous tetrahydrofuran(120 mL) at 0° C. is added diethylaluminum chloride (22.4 mL of a 1.8Msolution in toluene) over 20 minutes. The temperature is kept below 12°C. during the addition. The reaction is then allowed to stir at 0° C.for 90 minutes and then at room temperature for 4 hours. The reaction isthen cooled to 10° C. and quenched with saturated aqueous ammoniumchloride (200 mL). A 1M solution of sodium hydrogen tartrate (200 mL) isadded and the reaction is allowed to warm to room temperature. Thereaction is filtered and the solids rinsed with ethyl acetate. Thefiltrate layers are separated and the aqueous layer is extracted withethyl acetate. The combined organic layers are dried over anhydrousmagnesium sulfate, filtered and concentrated under vacuum. The residueis purified by flash chromatography (cyclohexane/ethyl acetate, 4:1) toprovide the title compound (8.34 g). The ratio of diastereomers isapproximately 1:1.

Preparation of4-tert-Butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenyl-N-(phenylmethyl)pentamide

Scheme I step B: To a solution of4-tert-butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenylpentanoicacid, ethyl ester (5.5 g, 11.5 mmol) in anhydrous tetrahydrofuran (50mL) is added at 0° C., benzylamine (6.15 g, 57.5 mmol). The reaction isstirred for 3 hours at 0° C., then at room temperature for 15 hours. Thereaction is then diluted with ethyl acetate (100 mL), washed with 0.1Naqueous hydrochloric acid (2×50 mL), water (50 mL), brine (50 mL) anddried over anhydrous magnesium sulfate. It is then filtered andconcentrated under vacuum. The residue is recrystallized from ethylacetate/pentane to provide the title compound (5.17 g) as a white solid.

Preparation of[3ξ,4(S)]-2,4,5-Trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]-amino]-2,2-difluoro-5-[4-(hydroxy)phenyl]-N-(phenylmethyl)-L-glycero-pentonamide

Scheme II step A; To a stirred suspension of Pd black (300 mg) in 4.4%HCO₂ H/CH₃ OH (25 mL) is added4-tert-butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenyl-N-benzylpentamide (6:1 R/S ratio, 1.39 g, 2.57 mmol). Additional 300 mg portionsof Pd black are added at 0.75 hours, 1.5 hours, and 2.25 hours. After4.25 hours total, the catalyst is removed by filtration (CH₃ OH rinse)and the filtrate is combined with that from a similar experiment (using51 mg of4-tert-butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenyl-N-benzylpentamide) and concentrated in vacuo. Recrystallization fromcyclohexane/EtOAc provides 1.10 g (92%) of the title compound(approximately 6:1 R/S ratio) as a fine ivory powder: mp 163-166° C.; IR(KBr) ν_(max) 3412, 3362, 1682, 1545, 1518, 1165 cm⁻¹ ; ¹ H NMR(DMSO-d₆) δ 9.18 (nm, 2 H), 7.35-7.2 (m, 5 H), 6.99 (d, 2 H, J=8.2 Hz),6.66 (d, 2 H, J=8.2 Hz), 6.19 (d, 1 H, J=9.1 Hz), 6.02 (d, 1 H, J=8.1Hz), 4.36 (dd, 1 H, J=15.5, 6.0 Hz), 4.27 (dd, 1 H, J=15.5, 6.2 Hz),4.0-3.87 (m, 2 H), 2.64 (m, 2 H), 1.33 (major) and 1.24 (2s, 9 H); ¹⁹ FNMR (DMSO-d₆) δ major diastereomer: -110.82 (dd, J=255, 6 Hz), -122.39(dd, J=255, 20 Hz), minor diastereomer: -111.05 (dd, J=255, 6 Hz),-121.78 (dd, J=255, 21 Hz); mass spectrum m/z 479 (M⁺ +29), 451 (M⁺ +1),423, 379, 352, 351 (100), 333, 243, 91.

Preparation of[3ξ,4(S)]-2,4,5-Trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide

Scheme II step B; To a stirred solution of[3ξ,4(S)]-2,4,5-Trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]-amino]-2,2-difluoro-5-[4-(hydroxy)phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(441 mg, 0.979 mmol) in acetone (6 mL) is added powdered K₂ CO₃ (165 mg,1.20 mmol), BrCH₂ CO₂ CH₃ (110 μL, 1.16 mmol), and a catalytic amount ofpowdered KI. The flask is stoppered and stirring is continued for 3days. The reaction mixture is poured into EtOAc/dilute aqueous NaCl, andthe organic layer is separated and washed with dilute aqueous KOH,brine, and dried over anhydrous magnesium sulfate. The organic layer isfiltered and concentrated under vacuum to provide 413 mg (81%) of thetitle compound as a tacky white solid. Recrystallization fromcyclohexane/EtOAc provides the title compound (5.5:1 R/S ratio) as awhite powder: mp 93.5-99.5° C.; IR (KBr) ν_(max) 3352, 1690, 1530, 1512,1215, 1177 cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.38-7.24 (m, 5H), 7.18 (nm, 1 H),7.10 (d, 2 H, J=8.6 Hz), 6.81 (d, 2 H, J=8.6 Hz), 5.00 (d, 1 H, J=9.2Hz), 4.72 (nm, 1 H), 4.60 and 4.58 (major) (2s in 1:5.5 ratio, 2 H),4.50 (dd, 1 H, J=14.8, 5.7 Hz), 4.42 (dd, 1 H, J=14.8, 5.7 Hz), 4.1-3.94(m, 2 H), 3.80 and 3.79 (major) (2s in 1:5.5 ratio, 3H), 3.0-2.8 (m, 2H), 1.42 and 1.38 (2s, 9 H); ¹⁹ F NMR (CDCl₃) δ minor diastereomer:-113.49 (dd, J=262, 9 Hz), major diastereomer: -115.83 (dd, J=262, 9 Hz;other F of minor diastereomer buried under this peak), -120.07 (dd,J=262, 14 Hz); mass spectrum, m/z 522 (M⁺), 495, 451, 423 (100), 405,243, 223, 91; [α]²⁰ _(D) -33.0° (c 0.81, CH₃ OH).

Preparation of[3ξ,4(S)]-2,4,5-Trideoxy-4-[[2-[[[[(1,1-dimethylethoxy)carbonyl]amino]acetyl]amino]-3-methyl-1-oxobutyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide

Scheme II steps C and D; A solution of[3ξ,4(S)]-2,4,5-trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(413 mg, 0.790 mmol) in trifluoroacetic acid (TFA) (4 mL) is allowed tostir under nitrogen for 2 hours. The solution is concentrated in vacuoand the residue is twice dissolved in EtOAc and concentrated again. Theresulting TFA salt is dissolved in 1:1 CH₂ Cl₂ /DMF (3 mL) with stirringunder nitrogen and 1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84mmol), N-methylmorpholine (NMM) (190 μL, 1.73 mmol), Boc-gly-val-OH (230mg, 0.84 mmol, prepared by reaction of commercially available gly-val-OHwith di-t-butyldicarbonate under standard conditions), and EDC (168 mg,0.88 mmol) are added in that order. After 3 days, the mixture is pouredinto water and extracted twice with EtOAc. The combined extracts arewashed with dilute aqueous HCl, NaHCO₃, and brine, and dried overanhydrous magnesium sulfate. The organic layer is concentrated undervacuum to provide 549 mg of gummy solid which is purified by flashchromatography (3:1 EtOAc/cyclohexane) to provide the title compound(443 mg) as a white solid. Recrystallization from EtOAc/cyclohexaneprovides the title compound (6.6:1 R/S ratio) as white granules: mp161-166° C.; IR (KBr) ν_(max) 3395, 3298, 1684, 1647, 1537, 1514, 1206,1179 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ 9.14 (nm, 1 H), 7.76 (d, 1 H, J=8.7 Hz),7.55 (d, 1 H, J=8.8 Hz), 7.35-7.2 (m, 5 H), 7.13 (d, 2 H, J=8.6 Hz),7.09 (m, 1 H), 6.84 (d, 2 H, J=8.6 Hz), 6.32 (d, 1 H, J=7.6 Hz), 4.75(major) and 4.73 (2s in 6.6:1 ratio, 2 H), 4.4-3.93 (m, 5 H), 3.69(major) and 3.69 (2s, 3 H), 3.56 (inner peaks of apparent AB, 2 H), 2.75(dd, 1 H, J=13.4, 8.1 Hz), 2.62 (dd, 1 H, J=13.4, 6.0 Hz), 1.98 (m, 1H), 1.38 (major) and 1.36 (2s, 9 H), 0.80 (d, 3 H, J=6.7 Hz), 0.76 (d, 3H, J=6.6 Hz); ¹⁹ F NMR (CDCl₃) δ major diastereomer: -110.67 (d, J=255Hz), -122.89 (dd, J=255, 20 Hz), minor diastereomer: -110.93 (d, J=257Hz), -122.29 (dd, J=257, 20 Hz); mass spectrum, m/z 707 (M⁺ +29), 679(M⁺ +1), 623, 579, 405 (100).

The pure [3(S),4(S)] title compound was obtained as a white powder afterrecrystallization from CH₃ OH/butanone/EtOAc: mp 209-211° C.; IR (KBr)ν_(max) 3306, 1680, 1653, 1537, 1514, 1211, 1179 cm⁻¹ ; ¹ H NMR(DMSO-d₆) δ (major rotamer) 9.25 (t, 1 H, J=6.0 Hz), 7.94 (d withupfield shoulder, 1 H, J=8.6 Hz), 7.41-7.21 (m, 6 H), 7.08-7.00 (m, 3H), 6.77 (d, 2 H, J=8.4 Hz), 6.25 (bs, 1 H), 4.72 (s, 2 H), 4.36 (m, 2H), 4.24-3.95 (m, 3 H), 3.69 (s, 3 H), 3.53 (inner peaks of apparent AB,not integrated), 2.94-2.81 (m, 1 H), 2.61 (dd, 1 H, J=14.1, 10.7 Hz),1.87 (m, 1 H), 1.38 (2s, 9 H), 0.72 (d, 3 H, J=7.0 Hz), 0.69 (d, 3 H,J=7.0 Hz); ¹⁹ F NMR (CDCl₃) δ -109.90 (dd, J=252, 7 Hz), -119.82 (dd,J=252, 19 Hz) [shoulders present at δ -109.8 and -119.9]; FAB massspectrum, m/z 679 (M⁺ +1), 579, 423, 405, 358, 307 (100), 289.

Preparation of[3ξ,4(S)]-2,4,5-Trideoxy-4-[[2-[[[[(1,1-dimethylethoxy)carbonyl]amino]acetyl]amino]-3-methyl-1-oxobutyl]amino]-2,2-difluoro-5-[4-[2-oxo-2-(pentafluorophenoxy)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide

Scheme II, steps E and F; To a stirred suspension of[3ξ,4(S)]-2,4,5-trideoxy-4-[[2-[[[[(1,1-dimethylethoxy)carbonyl]amino]acetyl]amino]-3-methyl-1-oxobutyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(400 mg, 0.589 mmol) in 19:1 CH₃ OH/H₂ O (20 mL) is added LiOH.H₂ O (29mg, 0.69 mmol). After 2 hours, additional LiOH.H₂ O (5 mg, 0.81 mmoltotal) is added, and after an additional 2 hours, the solution isconcentrated in vacuo. The residue is dissolved in water; the aqueoussolution is washed with ether, is covered with EtOAc, and is acidifiedwith vigorous stirring by the addition of 0.1 N NaHSO₄ (10 mL). Theorganic layer is separated, and the aqueous layer is extracted with asecond portion of EtOAc. The combined organic layers are washed withbrine and dried over anhydrous magnesium sulfate. The organic layer isconcentrated under vacuum to provide 407 mg (392 mg theory) of thecorresponding acid, which is dissolved in CH₂ Cl₂ (5 mL) and DMSO-d₆ (1mL). To this stirred solution under nitrogen is added C₆ F₅ OH (139 mg,0.755 mmol) and EDC (140 mg, 0.73 mmol). After 3 days the mixture isdiluted with water and filtered, washing the ivory solid with water andether. Attempted recrystallization from CF₃ CH₂ OH/EtOAc results inpartial transesterification to the trifluoroethyl ester. The mixture canbe saponified and reesterified to provide 394 mg of crude titlecompound. In a similar experiment recrystallization from CF₃ CH₂OH/EtOAc (filtering the hot solution through filter aid) also providespure title compound as fine white matted crystals: mp 202-204° C.; IR(KBr) ν_(max) 3389, 2974, 1684, 1653, 1522, 1173, 1121, 1080, 997 cm⁻¹ ;¹ H NMR (DMSO-d₆) δ 9.14 (m, 1 H), 7.77 (d, 1 H, J 9 Hz), 7.54 (d, 1 H,J=8.9 Hz), 7.35-7.22 (m, 5 H), 7.17 (d, 2 H, J=8.7 Hz), 7.08 (nm, 1 H),6.95 (d, 2 H, J=8.7 Hz), 6.33 (d, 1 H, J=7.6 Hz), 5.34 (s, 2 H),4.4-4.18 (m, 4 H), 4.08-3.95 (m, 1 H), 3.55 (nm, 2 H), 2.8-2.58 (m, 2H), 1.97 (m, 1 H), 1.38 (major) and 1.36 (2s, 9 H total), 0.80 (d, 3 H,J=6.6 Hz), 0.76 (d, 3 H, J=6.7 Hz); ¹⁹ F NMR (DMSO-d₆) δ -110.69 (d,J=256 Hz), -122.89 (dd, J=255, 20 Hz), -152.37 (d, J=20 Hz), -156.95 (t,J=23 Hz), -161.75 (dd, J=23, 20 Hz); mass spectrum, m/z 831 (M⁺ +1),775, 731. The [3(S),4(S)]-title compound is not isolated, but isconverted directly to the macrocyclic alcohol.

Preparation of[(9S),12(S)]-α,α-Difluoro-β-hydroxy-9-(1-methylethyl)-4,7,10-trioxo-N-(phenylmethyl)-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide

Scheme II, steps G and H;[3ξ,4(S)]-2,4,5-Trideoxy-4-[[2-[[[[(1,1-dimethylethoxy)carbonyl]amino]acetyl]amino]-3-methyl-1-oxobutyl]amino]-2,2-difluoro-5-[4-[2-oxo-2-(pentafluorophenoxy)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(494 mg, 0.595 mmol) is suspended in 4 N HCl/dioxane (16 mL) withstirring. After 2 hours, a clear gel forms. The solvent and HCl areremoved in vacuo and the residual solid is suspended in dilute aqueousNaHCO₃ /CH₂ Cl₂ with vigorous stirring for 3 days. The mixture isfiltered, and the ivory solids are washed with water and ether. HotEtOAc is added along with just enough CF₃ CH₂ OH to dissolve most of thesolids; filtration through filter aid and concentration under vacuumprovides 256 mg of title compound. In a similar experiment the filtrateis concentrated and diluted with hot EtOAc to obtain the (R)-alcohol ofthe title compound as fine white granules: mp >255° C.; IR (KBr) ν_(max)3412, 3318, 1663, 1537, 1514 cm⁻¹ ; ¹ H NMR (DMSO-d₆) δ 9.17 (m, 1 H),7.90 (m, 1 H), 7.64 (m, 1 H), 7.37-7.2 (m, 5 H), 7.11 (m, 1 H), 7.01 (m,1 H), 6.93 (m, 1 H), 6.81 (m, 1 H), 6.46 (m, 1 H), 6.14 (dd, 1 H, J=7.4,0.9 Hz), 4.60 ("d", 1 H, J=15 Hz), 4.5-4.1 (m, 6 H), 3.96 (m, 1 H),3.72-3.54 (2m, 2 H), 2.76 (m, 1 H), 1.78 (m, 1 H), 0.77-0.71 (m, 6 H);¹⁹ F NMR (DMSO-d₆) δ major conformer (85%) -109.96 (dd, J=256, 5 Hz),-122.71 (dd, J=256, 20 Hz), minor conformer (15%) -110.45 (d, traceimpurity),-122.3(m, trace impurity); mass spectrum, m/z 547 (M⁺ +1).

In the preparation of title compound, the crude material from thedeprotection/cyclization is triturated with several portions of boilingCH₃ OH to dissolve the (S)-alcohol of the title compound and remove someinsoluble polymeric material. The solvent is removed under vacuum andthe residue is triturated with several portions of boiling CF₃ CH₂ OH.The insoluble beige powder is the (S)-alcohol of the title compound: IR(KBr) ν_(max) 3401, 3298, 1678, 1643, 1543, 1514 cm⁻¹ ; ¹⁹ F NMR(DMSO-d₆) δ -108.94 (dd, J=253, 6 Hz), -121.27 (dd, J=253, 20 Hz); massspectrum, m/z 575 (M⁺ +29), 547 (M⁺ +1), 113 (100); exact ass calcd forC₂₇ H₃₃ F₂ N₄ O₆ 547.2368, found 547.2344. The (S)-alcohol of the titlecompound is not carried on in this particular experiment; however, itcan be subjected to the following reactions in a manner analogous to the(R)-alcohol to provide the ultimate title compound. In addition amixture of the (R) and (S)-alcohols can also be subjected to thefollowing reactions in an analgous manner to provide the ultimate finalproduct. The asymmetric center is destroyed in the final oxidation ofthis alcohol to provide the ketone, thus separation of these alcohols isnot critical.

Preparation of[(9S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-(phenylmethyl)-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide

Scheme II, step I;[(9S),12(S)]-α,α-Difluoro-β-hydroxy-9-(1-methylethyl)-4,7,10-trioxo-N-(phenylmethyl)-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide(240 mg, 0.439 mmol) is dissolved in DMSO (6 mL) by heating at 60° C.under nitrogen with vigorous stirring. Upon cooling, the solution isdiluted with CH₂ Cl₂ (2 mL) and cooled to -15 to -17° C. in an ice/CH₃OH bath. 2 M oxalyl chloride/CH₂ Cl₂ (2.0 mL) is added dropwise toprovide a thin slurry. After 1 hour, Et₃ N (1.15 mL, 8.25 mmol) is addedand the mixture is slowly allowed to warm to room temperature. After 17hours, the mixture is diluted with water/EtOAc. The organic layer isseparated and the aqueous layer is extracted with a second portion ofEtOAc; some insoluble white solid (18 mg) is present which is startingmaterial. The combined organic extracts are washed three times withwater, brine, and dried over anhydrous magnesium sulfate. The organiclayer is then filtered and concentrated under vacuum. The crude whiteresidue (101 mg) is purified by flash chromatography (19:1 EtOAc/CH₃ OH)to provide 27 mg of a nonpolar oil (discarded) and 70 mg of a whitesolid/gel. Repeated recrystallizations from EtOAc/CF₃ CH₂ OH gave awhite gel which is washed with 19:1 CH₂ Cl₂ /CH₃ OH to give 17 mg of thetitle compound as a light beige powder, a mixture consisting primarilyof the minor [9(S),12(R)] diastereomer, but also containing the[9(S),12(S)] diastereomer, as well as a presumed hydrate of the[9(S),12(R)] diastereomer. The insoluble gel was recrystallized furtherfrom the same solvent mixture to give 5 mg of the [9(S),12(S)]diasteromer as fine, light beige granules. For the mixture: IR (KBr)ν_(max) 3420, 1669, 1530, 1514 cm⁻¹ ; ¹⁹ F NMR (DMSO-d₆) δ [9(S),12(R)]diastereomer: -105.89 (d, J=263 Hz), -111.90 (d, J=263 Hz); [9(S),12(S)]diastereomer: -109.13 (d, J=274 Hz) -111.77 (d, J=274 Hz); presumedhydrate of the [9(S),12(R)] diastereomer: -105.62 (d, J=271 Hz), -123.35(d, J=271 Hz) (70:18:12 mixture, respectively); mass spectrum (CI, 70eV), m/z 573 (M⁺ +29), 571, 545 (M⁺ +1), 308, 268, 250 (100), 190, 91;exact mass calcd for C₂₇ H₃₀ F₂ N₄ O₆ 545.2212, found 545.2239. For the[9(S),12(S)] diastereomer: IR (KBr) ν_(max) 3418, 1667, 1535, 1514 cm⁻¹; ¹⁹ F NMR (DMSO-d₆) δ -109.12 (d, J=274 Hz); -111.77 (d, J=274 Hz),plus minor impurities; mass spectrum (CI, 70 eV), m/z 573 (M⁺ +29), 545(M⁺ +1), 308 (100), 91; exact mass calcd for C₂₇ H₃₀ F₂ N₄ O₆ 545.2212,found 545.2230.

EXAMPLE 2 Preparation of[(9S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-[2-methyl-1-[(phenylmethoxy)methyl]propyl]-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide##STR22## Preparation of the starting material O-benzyl-D-valinolrequired for the following reaction

A solution of D-valinol (5.1 g, 49.4 mmol) and di-tert-butyldicarbonate(10.9 g, 52 mmol) in methanol (60 mL) is stirred for 17 hours at roomtemperature. After concentration under vacuum, the residue is purifiedby flash chromatography (silica gel, ethyl acetate/petroleum ether: 3/7,R_(f) : 0.37) to provide N-tert-butoxycarbonyl-D-valinol in quantitativeyield (10.07 g, colorless oil).

To a solution of N-tert-butoxycarbonyl-D-valinol (10 g, 49.3 mmol) andbenzylbromide (5.86 mL, 49.3 mmol) in anhydrous dimethyl formamide (50mL) is added at -5° C. and under nitrogen, potassium-tert-butoxide(11.06 g, 98.6 mmol) as a solid, portionwise, an in such a way that theinternal temperature does not exceed +5° C. The reaction mixture isstirred for 2 hours at 0° C., diluted with ethyl acetate (2×300 mL),extracted with a 1N solution of potassium hydrogen sulfate (50 mL) andwater (250 mL) and is washed twice with water (2×200 mL). After dryingof the organic phase over anhydrous sodium sulfate, filtration andconcentration under vacuum, the oil is purified by flash chromatography(silica gel, ethyl acetate/petroleum ether: 1/9, Rf: 0.42) to provideN-tert-butoxycarbonyl-O-benzyl-D-valinol as a colorless oil (9.95 g, 69%yield).

A solution of N-tert-butoxycarbonyl-O-benzyl-D-valinol (9.95 g, 34 mmol)in formic acid (50 mL) is stirred for 4 hours at room temperature. Afterremoval of the formic acid under vacuum, the sticky residue is dissolvedin water (100 mL), neutralized with a saturated solution of sodiumbicarbonate (100 mL) and the organic material is extracted twice withethyl acetate (2×200 mL). The organic phases are washed until neutralwith water (2×200 mL) and the combined organic layers are dried overanhydrous sodium sulfate. Filtration and concentration under vacuumprovides O-benzyl-D-valinol as a slightly yellowish oil (5.20 g, 79%).

Scheme I step C; Combine4-tert-butoxycarbonylamino-2,2-difluoro-3-hydroxy-5-(4-benzyloxy)phenylpentanoicacid, ethyl ester (756 mg, 1.58 mmol, prepared in example 1, Scheme Istep A) and 4.4% formic acid/methanol (9 mL) under an atmosphere ofnitrogen. Add palladium black (171 mg) and stir for 1 hour. After 1hour, then 4 hours and finally after 2 days, add respectively additionalamounts of palladium black (80 mg, 378 mg and 111 mg). After 6 daysfilter the reaction and concentrate the filtrate under vacuum. Purifythe residue by flash chromatography (silica gel, cyclohexane/ethylacetate, 2:1 followed by 1:1) to provide the debenzylated product (380mg, 58%) as a light yellow foam.

Scheme I step D; Add trimethylaluminum (1.55 mL of a 2M solution intoluene) dropwise to a solution of O-benzyl-D-valinol (600 mg, 3.11mmol, prepared above) in dry dichloromethane (1 mL) under an atmosphereof nitrogen. Stir the reaction for 15 minutes and add a solution of theabove prepared debenzylated product (380 mg, 0.976 mmol) in drydichloromethane (1 mL). Add an additional amount of dichloromethane (3mL) and stir for 19 hours at room temperature. Add dry tetrahydrofuran(5 mL) and stir for 3 hours. Partition the reaction between cold diluteaqueous hydrochloric acid and ethyl acetate. Separate the layers andwash the organic layer with water and brine. Dry the organic layer overanhydrous magnesium sulfate, filter and concentrate under vacuum.Subject the residue to a second amidation reaction under identicalconditions as above to drive the reaction further toward completion.Work up the second reaction in a manner analogous to the first reaction.Purify the residue by flash chromatography (silica gel,cyclohexane/ethyl acetate, 5:3) to provide impure product (351 mg) whichis contaminated with ester starting material. To purify the productfurther dissolve the above impure product in methanol (10 mL) and water(0.5 mL). Add lithium hydroxide.H₂ O (48 mg) and stir for 3 hours. Thenpartially concentrate the reaction under vacuum, dilute with water, addether and cold dilute aqueous hydrochloric acid. Separate the layers andextract the aqueous with ether. Combine the organic layer and extractand wash with water, aqueous potassium carbonate (2×) and brine. Dry theorganic layers over anhydrous magnesium sulfate, filter and concentrateunder vacuum to provide the amide (3.5:1 R/S at the hydroxyl) (266 mg,51%): ¹⁹ F NMR (CDCl₃) δ (R) diastereomer: -115.57 (dd, J=260, 8 Hz),-121.65 (dd, J=260, 17 Hz); (S) diastereomer: -112.00 (d, J=266 Hz),-120.7 (dd, J=266, 18 Hz).

Scheme II step B; Combine the above prepare amide (266 mg, 0.496 mmol)with powdered potassium carbonate (80 mg, 0.58 mmol) in acetone (3 mL)under an atmosphere of nitrogen. Add dropwise to the stirring mixturemethyl bromoacetate (56 μL, 0.59 mmol). Stir the reaction for 3 days.The product is worked-up in a manner analogous to that described inexample 1, Scheme II step B. If residual starting material remainssubject the impure product to the same alkylating conditions asdescribed above with catalytic amount of potassium iodide added. Stirfor 24 hours. Isolate the product by the work up procedure describedpreviously. Purify by flash chromatography (silica gel,cyclohexane/ethyl acetate, 5:3) to provide the desired alkylated product(143 mg, 47%, 5:1 R/S at the hydroxyl): ¹⁹ F NMR (CDCl₃) δ (R)diastereomer: -155.53 (dd, J=261, 7 Hz), -122.06 (dd, J=261, 17 Hz); (S)diastereomer: -113.83 (d, J=256 Hz), -127.56 (dd, J=256, 19 Hz).

Scheme II step C; Combine the above prepared alkylated product (143 mg,0.235 mmol) with formic acid (3 mL, 96%) and stir the reaction at roomtemperature for 1.5 hours. Concentrate the reaction under vacuum andpartition the residue between ethyl acetate and dilute aqueous sodiumbicarbonate. Separate the organic layer and wash with water (2×).Concentrate under vacuum to provide the deprotected amine (114 mg, 95%).

Scheme II step D; Combine the above deprotected amine (114 mg),1-hydroxybenzotriazole hydrate (38 mg, 0.25 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (55 mg, 0.29mmol), N-methylmorpholine (28 μL, 0.25 mmol) and Boc.gly.val (69 mg,0.25 mmol) in dichloromethane/dimethylformamide at 0° C. The reaction isallowed to warm to room temperature and after 16 hours, the mixture ispoured into water and extracted twice with EtOAc. The combined extractsare washed with dilute aqueous HCl, NaHCO₃, and brine, and dried overanhydrous magnesium sulfate. The organic layer is filtered andconcentrated under vacuum. The residue is purified by flashchromatography (silica gel, ethyl acetate/cyclohexane, 70:30) followedby recrystallization from cyclohexane/ethyl acetate to provide thedesired amide (137 mg, 76%) as fine white granules: mp 161.5-163.5° C.;IR (KBr) ν_(max) 1696, 1653, 1514 cm⁻¹ ; ¹ H NMR (CDCl₃) δ 7.37-7.26 (m,5H), 7.11(d, 2H, J=8.6 Hz), 6.9 (m, 2H), 6.78 (d, 2H, J=8.6 Hz), 6.62(bd, 1H), 5.44 (m, 1H), 4.86 (bs, 1H), 4.60 (s, 2H), 4.54 (d, 1H, J=11.9Hz), 4.46 (d, 1H, J=11.9 Hz), 4.4-4.3 (m, 1H), 4.2-4.03 (m, 2H),3.9-3.75 (m, 2H), 3.81 (s, 3H), 3.67-3.59 (m, 2H), 3.49 (dd, 1H, J=10.0,3.8 Hz), 2.89 (apparent doublet, 2H), 2.15-1.92 (m, 2H), 1.45 (s, 9H),0.94 (d, 6H, J=6.75 Hz), 0.88 (d, 3H, J=6.7 Hz), 0.84 (d, 3H, J=6.6 Hz);¹⁹ F NMR (CDCl₃) δ -116.78 (d, J=258 Hz), -120.16 (dd, J=258, 9 Hz);mass spectrum (FAB), m/z 765 (M⁺ +1), 709, 665, 509(100), 419, 382.

Scheme II steps E and F; Combine the above prepared amide (137 mg, 0.179mmol) with lithium hydroxide .H₂ O (12 mg, 0.29 mmol) in methanol (4.5mL) and water (0.5 mL). Stir the reaction for 3 hours. The reaction isthen concentrated under vacuum. The residue is dissolved in water. Theaqueous solution is washed with ether, is covered with EtOAc, and isacidified with vigorous stirring by the addition of 0.1 N NaHSO₄. Theorganic layer is separated, and the aqueous layer is extracted with asecond portion of EtOAc. The combined organic layers are washed withbrine and dried over anhydrous magnesium sulfate. The organic layer isconcentrated under vacuum to provide the corresponding acid which isdissolved in CH₂ Cl₂ (3 mL). To this stirred solution under nitrogen isadded C₆ F₅ OH (40 μL,, 0.35 mmol) and EDC (45 mg, 0.23 mmol). After 1day the mixture is diluted with water and filtered to provide thedesired pentafluorophenyl ester (161 mg, 98%) as fine white granules.Recrystallization from cyclohexane/ethyl acetate provides thepentafluorophenyl ester; mp 139.5-143° C.; IR (KBr) ν_(max) 3416, 3376,3312, 1697, 1661, 1522, 1171, 1121, 1078, 997 cm⁻¹ ; ¹ H NMR (CDCl₃) δ7.39-7.25 (m, 5H), 7.16 (d, 2H, J=8.5 Hz), 6.94-6.78 (m, 2H), 6.86 (d,2H, J=8.6 Hz), 6.57 (d, 1H, J=9.0 Hz), 5.24 (nm, 1H), 4.96 (s, 2H), 4.74(nm, 1H), 4.54 (d, 1H, J=11.9 Hz), 4.45 (d, 1H, J=11.9 Hz), 4.30 (m,1H), 4.19-3.99 (m, 2H), 3.9-3.79 (m, 1H), 3.73 (dd, 1H, J=18.2, 5.6 Hz),3.64 (m, 2H), 3.49 (m, 1H), 2.91 (apparent narrow d, 2H), 2.11 (m, 1H),1.99 (m, 1H), 1.45 (s, 9H), 0.94 (d, 3H, J=6.7 Hz), 0.93 (d, 3H, J=6.75Hz), 0.89 (d, 3H, J=6.75 Hz), 0.84 (d, 3H, J=6.95 Hz); ¹⁹ F NMR (CDCl₃)δ -116.65 (d, 1F, J=259 Hz), -120.28 (dd, 1F, J=262, 9 Hz), -152.68 (d,2F, J=18 Hz), -157.39 (t, 1F, J=22 Hz), -162.13 (dd, 2F, J=22, 18 Hz);mass spectrum (FAB), m/z 917(M⁺ +1), 861, 817, 661(100), 571, 534, 360,331, 173.

Scheme II steps G and H; Combine the above prepared pentafluorophenylester (155 mg, 0.169 mmol) with formic acid (4.5 mL, 96%) and stir for 2hours. Concentrate the reaction under vacuum. Add methylene chloride (50mL) and saturated sodium bicarbonate (50 mL). Stir the reaction for 3days. Add ethyl acetate and filter through fine fritted glass filter.Wash the gel with water. Separate the organic layer in the filtrate,wash with water (3×) and concentrate under vacuum. Purify the residue byflash chromatography (silica gel, 95% methanol/ethyl acetate) to providethe macrocyclic alcohol (9 mg, 8% (R) diastereomer at the hydroxyl) as awaxy white solid: ¹ H NMR (CD₃ OD) δ 7.42-7.27 (m, 5H), 7.23 (m, 1H),7.00 (m, 1H), 6.91 (m, 1H), 6.58 (m, 1H), 4.71 (d, 1H, J=16.0 Hz),4.65-4.5 (m, 1H), 4.59 (d,1H, J=12.1 Hz), 4.56 (d, 1H, J=15.6 Hz), 4.54(d, 1H, J=12.1 Hz), 4.24 (m, 1H), 4.08-4.01 (m, 2H), 3.94 (narrow m,1H), 3.70-3.57 (m, 3H), 2.93 (dd, 1H, J=13.2, 3.4 Hz), 2.73 (dd, 1H,J=13.1, 12.5 Hz), 2.01 (m, 1H), 1.89 (m, 1H), 1.01 (d, 3H, J=6.2 Hz),0.99 (d, 3H, J=5.9 Hz), 0.91 (d, 3H, J=6.9 Hz), 0.87 (d, 3H, J=6.8 Hz);¹⁹ F NMR (CD₃ OD) δ -114.75 (dd, J=258, 9 Hz), -121.97 (dd, J=258, 17Hz).

Scheme II step I; To a stirred solution of the above preparedmacrocyclic alcohol (9 mg, 0.014 mmol) in 1:1 methylenechloride/acetonitrile (8 mL) under nitrogen is added the Dess-Martinperiodinane (30 mg, 0.071 mmol). The resulting suspension is allowed tostir at room temperature for 3 days. The mixture is then diluted withethyl acetate/aqueous sodium bicarbonate and sodium thiosulfate. After10 minutes, the organic layer is separated, washed with water andconcentrated under vacuum to provide a mixture of recovered alcohol,ketone and ketone hydrate. The mixture is resubjected to the oxidationreaction using periodinane (30 mg, 0.071 mmol) in 3:1acetonitrile/methylene chloride (4 mL). After 7 days, the mixture isworked up as above to provide a mixture of the title compound and thehydrate of the title compound (6 mg total) as a white solid: ¹⁹ F NMR(CD₃ CN) δ ketone: -111.93 and -111.96 (2s, inner peaks of an ABpattern), ketone hydrate: -115.36 (d, J=257 Hz), -119.02 (d, J=257 Hz).

EXAMPLE 3 Preparation ofN-Benzyl-3-(6-benzyl-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl)-2,2-difluoro-3-oxo-propionamide##STR23##

Scheme II steps C and D; A solution of[3ξ,4(S)]-2,4,5-trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(0.790 mmol, prepared in example 1 Scheme II step B) in trifluoroaceticacid (TFA) (4 mL) is allowed to stir under nitrogen for 2 hours. Thesolution is concentrated in vacuo and the residue is twice dissolved inEtOAc and concentrated again. The resulting TFA salt is dissolved in 1:1CH₂ Cl₂ /DMF (3 mL) with stirring under nitrogen and1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (190 μL, 1.73 mmol), Boc-phe-val-OH (0.84mmol), and EDC (168 mg, 0.88 mmol) are added in that order. After 3days, the mixture is poured into water and extracted twice with EtOAc.The combined extracts are washed with dilute aqueous HCl, NaHCO₃, andbrine, and dried over anhydrous magnesium sulfate. The organic layer isconcentrated under vacuum to provide the desired amide.

Scheme II steps E and F; To a stirred suspension of the above preparedamide (0.589 mmol) in 19:1 CH₃ OH/H₂ O (20 mL) is added LiOH.H₂ O (34mg, 0.81 mmol). After 2 hours, the solution is concentrated in vacuo.The residue is dissolved in water; the aqueous solution is washed withether, is covered with EtOAc, and is acidified with vigorous stirring bythe addition of 0.1 N NaHSO₄ (10 mL). The organic layer is separated,and the aqueous layer is extracted with a second portion of EtOAc. Thecombined organic layers are washed with brine and dried over anhydrousmagnesium sulfate. The organic layer is concentrated under vacuum toprovide the corresponding acid, which is dissolved in CH₂ Cl₂ (5 mL). Tothis stirred solution under nitrogen is added C₆ F₅ OH (139 mg, 0.755mmol) and EDC (140 mg, 0.73 mmol). After 1 day the mixture is dilutedwith water and filtered, washing the solid with water and ether toprovide the desired pentafluorophenyl ester. Alternatively the desiredpentafluorophenyl ester can be isolated by extractive methods well knownin the art.

Scheme II steps G and H: The above prepared pentafluorophenyl ester issuspended in 4 N HCl/dioxane (16 mL) with stirring. After 2 hours thesolvent and HCl are removed in vacuo and the residual solid/gel issuspended in dilute aqueous NaHCO₃ /CH₂ Cl₂ with vigorous stirring for 3days. The mixture is filtered, and the solids are washed with water andether. Hot EtOAc is added along with just enough CF₃ CH₂ OH to dissolvemost of the solids; filtration through filter aid and concentrationunder vacuum provides the desired macrocyclic alcohol.

Scheme II step I: To a stirred solution of the above preparedmacrocyclic alcohol (0.014 mmol) in 1:1 methylene chloride/acetonitrile(8 mL) under nitrogen is added the Dess-Martin periodinane (60 mg, 0.14mmol). The resulting suspension is allowed to stir at room temperaturefor 3 days. The mixture is then diluted with ethyl acetate/aqueoussodium bicarbonate, sodium thiosulfate. After 10 minutes, the organiclayer is separated, washed with water and concentrated under vacuum toprovide the title compound.

EXAMPLE 4 Preparation of3-[12-(Benzylcarbamoyl-difluoro-acetyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-6-yl]-propionicacid ##STR24##

Scheme II step C; A solution of[3ξ,4(S)]-2,4,5-trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(0.790 mmol, prepared in example 1 Scheme II step B) in trifluoroaceticacid (TFA) (4 mL) is allowed to stir under nitrogen for 2 hours. Thesolution is concentrated in vacuo and the residue is twice dissolved inEtOAc and concentrated again to provide the TFA salt of the deprotectedamine.

Scheme III step A; The TFA salt of the deprotected amine prepared aboveis dissolved in 1:1 CH₂ Cl₂ /DMF (3 mL) with stirring under nitrogen and1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (190 μL, 1.73 mmol), Boc-val-OH (0.84 mmol),and EDC (168 mg, 0.88 mmol) are added in that order. After 3 days, themixture is poured into water and extracted twice with EtOAc. Thecombined extracts are washed with dilute aqueous HCl, NaHCO₃, and brine,and dried over anhydrous magnesium sulfate. The organic layer isconcentrated under vacuum to provide the desired amide.

Scheme III step B and C; A solution of the above prepared amide (0.790mmol) in trifluoroacetic acid (TFA) (4 mL) is allowed to stir undernitrogen for 2 hours. The solution is concentrated in vacuo and theresidue is twice dissolved in EtOAc and concentrated again to providethe TFA salt of the deprotected amine. The TFA salt of the deprotectedamine is dissolved in 1:1 CH₂ Cl₂ /DMF (3 mL) with stirring undernitrogen and 1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (190 μL, 1.73 mmol), Nα-FMOC-γ-tert-butylester-glu-OH (0.84 mmol), and EDC (168 mg, 0.88 mmol) are added in thatorder. After 3 days, the mixture is poured into water and extractedtwice with EtOAc. The combined extracts are washed with dilute aqueousHCl, NaHCO₃, and brine, and dried over anhydrous magnesium sulfate. Theorganic layer is concentrated under vacuum to provide the desired amide.

Alternative method for cyclization

The above prepared amide (0.6 mmol) is dissolved in methanol/water(19:1) and lithium hydroxide.H₂ O (1.2 mmol) is added with stirring.After 5 hours the reaction is diluted with water and rinsed with ether.The aqueous layer is then acidified to pH 4.5-5 with 0.1N aqueous sodiumbisulfate. The acidified aqueous layer is then extracted with ethylacetate. The organic layer is dried over anhydrous magnesium sulfate,filtered and concentrated under vacuum to provide the desiredacid/deprotected amine as shown below. ##STR25##

The above prepared acid/deprotected amine (0.70 mmol) is dissolved in1:1 CH₂ Cl₂ /DMF (3 mL) with stirring under nitrogen and1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (95 μL, 0.87 mmol) and EDC (168 mg, 0.88 mmol)are added in that order. After 3 days, the mixture is poured into waterand extracted twice with EtOAc. The combined extracts are washed withdilute aqueous HCl, NaHCO₃, and brine, and dried over anhydrousmagnesium sulfate. The organic layer is concentrated under vacuum toprovide the macrocyclic alcohol.

Scheme II step I; To a stirred solution of the above preparedmacrocyclic alcohol (0.014 mmol) in 1:1 methylene chloride/acetonitrile(8 mL) under nitrogen is added the Dess-Martin periodinane (60 mg, 0.14mmol). The resulting suspension is allowed to stir at room temperaturefor 3 days. The mixture is then diluted with ethyl acetate/aqueoussodium bicarbonate, sodium thiosulfate. After 10 minutes, the organiclayer is separated, washed with water and concentrated under vacuum toprovide the ketone.

Scheme II step J; Dissolve the above prepared ketone (0.013 mmol) inmethylene chloride (4 mL) and add trifluoroacetic acid (1 mL). Stir thereaction for 3 hours at room temperature and then concentrate undervacuum to provide the title compound.

EXAMPLE 5 Preparation of3-[6-(4-Amino-butyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl]-N-benzyl-2,2-difluoro-3-oxo-propionamide##STR26##

Scheme II steps C and D; A solution of[3ξ,4(S)]-2,4,5-trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(0.790 mmol, prepared in example 1 Scheme II step B) in trifluoroaceticacid (TFA) (4 mL) is allowed to stir under nitrogen for 2 hours. Thesolution is concentrated in vacuo and the residue is twice dissolved inEtOAc and concentrated again. The resulting TFA salt is dissolved in 1:1CH₂ Cl₂ /DMF (3 mL) with stirring under nitrogen and1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (190 μL, 1.73 mmol),Nα-t-Boc-Nε-Cbz-L-lys-val-OH (0.84 mmol), and EDC (168 mg, 0.88 mmol)are added in that order. After 1 day, the mixture is poured into waterand extracted twice with EtOAc. The combined extracts are washed withdilute aqueous HCl, NaHCO₃, and brine, and dried over anhydrousmagnesium sulfate. The organic layer is concentrated under vacuum toprovide the desired amide.

Scheme II steps E and F; To a stirred suspension of the above prepareamide (0.589 mmol) in 19:1 CH₃ OH/H₂ O (20 mL) is added LiOH.H₂ O (34mg, 0.81 mmol). After 2 hours, the solution is concentrated in vacuo.The residue is dissolved in water; the aqueous solution is washed withether, is covered with EtOAc, and is acidified with vigorous stirring bythe addition of 0.1 N NaHSO₄ (10 mL). The organic layer is separated,and the aqueous layer is extracted with a second portion of EtOAc. Thecombined organic layers are washed with brine and dried over anhydrousmagnesium sulfate. The organic layer is concentrated under vacuum toprovide the corresponding acid, which is dissolved in CH₂ Cl₂ (5 mL). Tothis stirred solution under nitrogen is added C₆ F₅ OH (139 mg, 0.755mmol) and EDC (140 mg, 0.73 mmol). After 1 day the mixture is dilutedwith water and filtered, washing the solid with water and ether toprovide the desired pentafluorophenyl ester. Alternatively the desiredpentafluorophenyl ester can be isolated by extractive methods well knownin the art.

Scheme II steps G and H: The above prepared pentafluorophenyl ester issuspended in 4 N HCl/dioxane (16 mL) with stirring. After 2 hours thesolvent and HCl are removed in vacuo and the residual solid/gel issuspended in dilute aqueous NaHCO₃ /CH₂ Cl₂ with vigorous stirring for 3days. The mixture is filtered, and the solids are washed with water andether. Hot EtOAc is added along with just enough CF₃ CH₂ OH to dissolvemost of the solids; filtration through filter aid and concentrationunder vacuum provides the desired macrocyclic alcohol.

Scheme II step I: To a stirred solution of the above preparedmacrocyclic alcohol (0.014 mmol) in 1:1 methylene chloride/acetonitrile(8 mL) under nitrogen is added the Dess-Martin periodinane (60 mg, 0.14mmol). The resulting suspension is allowed to stir at room temperaturefor 3 days. The mixture is then diluted with ethyl acetate/aqueoussodium bicarbonate, sodium thiosulfate. After 10 minutes, the organiclayer is separated, washed with water and concentrated under vacuum toprovide the desired ketone.

Scheme II step J; To a stirred suspension of Pd black (10 mg) in 4.4%HCO₂ H/methanol (5 mL) is added the above prepared ketone (0.014 mmol).After 4 hours the reaction is filtered and the filtrate is concentratedunder vacuum to provide the title compound.

EXAMPLE 6 Preparation ofN-Benzyl-3-[6-(2-carbamoylethyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl]-2,2-difluoro-3-oxo-propionamide##STR27##

Scheme II steps C and D; A solution of[3ξ,4(S)]-2,4,5-trideoxy-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2,2-difluoro-5-[4-[2-methoxy-2-(oxo)ethoxy]phenyl]-N-(phenylmethyl)-L-glycero-pentonamide(0.790 mmol, prepared in example 1 Scheme II step B) in trifluoroaceticacid (TFA) (4 mL) is allowed to stir under nitrogen for 2 hours. Thesolution is concentrated in vacuo and the residue is twice dissolved inEtOAc and concentrated again. The resulting TFA salt is dissolved in 1:1CH₂ Cl₂ /DMF (3 mL) with stirring under nitrogen and1-hydroxybenzotriazole hydrate (HOBT) (128 mg, 0.84 mmol),N-methylmorpholine (NMM) (190 μL, 1.73 mmol), Boc-gln-val-OH (0.84mmol), and EDC (168 mg, 0.88 mmol) are added in that order. After 1 day,the mixture is poured into water and extracted twice with EtOAc. Thecombined extracts are washed with dilute aqueous HCl, NaHCO₃, and brine,and dried over anhydrous magnesium sulfate. The organic layer isconcentrated under vacuum to provide the desired amide.

Scheme II steps E and F; To a stirred suspension of the above prepareamide (0.589 mmol) in 19:1 CH₃ OH/H₂ O (20 mL) is added LiOH.H₂ O (34mg, 0.81 mmol). After 2 hours, the solution is concentrated in vacuo.The residue is dissolved in water; the aqueous solution is washed withether, is covered with EtOAc, and is acidified with vigorous stirring bythe addition of 0.1 N NaHSO₄ (10 mL). The organic layer is separated,and the aqueous layer is extracted with a second portion of EtOAc. Thecombined organic layers are washed with brine and dried over anhydrousmagnesium sulfate. The organic layer is concentrated under vacuum toprovide the corresponding acid, which is dissolved in CH₂ Cl₂ (5 mL). Tothis stirred solution under nitrogen is added C₆ F₅ OH (139 mg, 0.755mmol) and EDC (140 mg, 0.73 mmol). After 1 day the mixture is dilutedwith water and filtered, washing the solid with water and ether toprovide the desired pentafluorophenyl ester. Alternatively the desiredpentafluorophenyl ester can be isolated by extractive methods well knownin the art.

Scheme II steps G and H: The above prepared pentafluorophenyl ester issuspended in 4 N HCl/dioxane (16 mL) with stirring. After 2 hours thesolvent and HCl are removed in vacuo and the residual solid/gel issuspended in dilute aqueous NaHCO₃ /CH₂ Cl₂ with vigorous stirring for 3days. The mixture is filtered, and the solids are washed with water andether. Hot EtOAc is added along with just enough CF₃ CH₂ OH to dissolvemost of the solids; filtration through filter aid and concentrationunder vacuum provides the desired macrocyclic alcohol.

Scheme II step I: To a stirred solution of the above preparedmacrocyclic alcohol (0.014 mmol) in 1:1 methylene chloride/acetonitrile(8 mL) under nitrogen is added the Dess-Martin periodinane (60 mg, 0.14mmol). The resulting suspension is allowed to stir at room temperaturefor 3 days. The mixture is then diluted with ethyl acetate/aqueoussodium bicarbonate, sodium thiosulfate. After 10 minutes, the organiclayer is separated, washed with water and concentrated under vacuum toprovide the title compound.

In a further embodiment the present invention provides a method oftreating a patient afflicted with a viral infection comprising theadministration thereto of an effective antiviral amount of a compound offormula (I).

The term "viral infection" as used herein refers to an abnormal state orcondition characterized by viral transformation of cells, viralreplication and proliferation. Viral infections for which treatment witha compound of formula (I) will be particularly useful includeretroviruses such as but not limited to HTLV-I, HTLV-II, HTLV-III (HIVvirus), murine leukemia virus, feline leukemia virus,cytomegalovirus(CMV), avian sarcoma virus and the like. In additiontreatment with a compound of formula (I) would be useful in treating awide range of states of HIV infection: AIDS, ARC (AIDS related complex),both symptomatic and asymptomatic, and actual or potential exposure toHIV. For example, compounds of this invention are useful in treatingsymptomatic AIDS after suspected past exposure to HIV by, e.g., bloodtransfusion, accidental needle stick, or exposure to patient bloodduring surgery.

An "effective antiviral amount" of a compound of formula (I) refers toan amount which is effective, upon single or multiple doseadministration to the patient, in controlling the growth of the virus orin prolonging the survivability of the patient beyond that expected inthe absence of such treatment. As used herein "controlling a viralinfection" refers to slowing, interrupting, arresting or stopping theviral transformation of cells or the replication and proliferation ofthe virus and does not necessarily indicate a total elimination of thevirus.

The present invention further provides a method of inhibiting HIVprotease in a patient in need thereof comprising administering to saidpatient an effective inhibitory amount of a compound of formula (I).

It is understood that patients suffering from a retrovirus, such asHTLV-III are in need of an HIV protease inhibitor such as a compound offormula (I).

As used herein, the term "patient" refers to a warm-blooded animal, suchas a mammal, which is afflicted with a particular viral infection. It isunderstood that humans, mice and rats are included within the scope ofthe term "patient".

Administration of a compound of formula (I) to a patient results ininhibition of HIV protease in the patient. Thus, by treatment of apatient with a compound of formula (I) retroviruses, such as HTLV-III,are inhibited or suppressed.

A patient is in need of treatment with an agent which inhibits HIVprotease, such as a compound of formula (I), where the patient issuffering from certain viral infections for which HIV protease isimplicated as a contributing factor in the progression of the disease.

Based on standard clinical and laboratory tests and procedures, anattending diagnostician, as a person skilled in the art, can readilyidentify those patients who are in need of treatment with an agent whichinhibits HIV protease, such as a compound of formula (I).

An "effective inhibitory amount" of a compound of formula (I) is thatamount which is effective, upon single or multiple does administrationto a patient, in providing an inhibition of HIV protease.

As used herein the term "effective amount" refers to an effectiveantiviral or inhibitory amount of a compound of formula (I). Aneffective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose, a number of factors areconsidered by the attending diagnostician, including, but not limitedto: the species of mammal; its size, age, and general health; thespecific viral infection involved; the degree of or involvement or theseverity of the viral infection; the response of the individual patient;the particular compound administered; the mode of administration; thebioavailability characteristics of the preparation administered; thedose regimen selected; the use of concomitant medication; and otherrelevant circumstances.

An effective amount of a compound of formula (I) is expected to varyfrom about 0.1 milligram per kilogram of body weight per day (mg/kg/day)to about 100 mg/kg/day. Preferred amounts are expected to vary fromabout 0.5 to about 10 mg/kg/day.

In effecting treatment of a patient afflicted with a viral infection, acompound of formula (I) can be administered in any form or mode whichmakes the compound bioavailable in effective amounts, including oral andparenteral routes. For example, compounds of formula (I) can beadministered orally, subcutaneously, intramuscularly, intravenously,transdermally, intranasally, rectally, and the like. Oral administrationis generally preferred. One skilled in the art of preparing formulationscan readily select the proper form and mode of administration dependingupon the particular characteristics of the compound selected, the viralinfection to be treated, the stage of the infection, and other relevantcircumstances.

The compounds of formula (I) can be administered alone or in the form ofa pharmaceutical composition in combination with pharmaceuticallyacceptable carriers or excipients, the proportion and nature of whichare determined by the solubility and chemical properties of the compoundselected, the chosen route of administration, and standardpharmaceutical practice. The compounds of the invention, while effectivethemselves, may be formulated and administered in the form of theirpharmaceutically acceptable salts for purposes of stability, convenienceof crystallization, increased solubility and the like.

In another embodiment, the present invention provides compositionscomprising a compound of formula (I) in admixture or otherwise inassociation with one or more inert carriers. These compositions areuseful, for example, as assay standards, as convenient means of makingbulk shipments, or as pharmaceutical compositions. An assayable amountof a compound of formula (I) is an amount which is readily measurable bystandard assay procedures and techniques as are well known andappreciated by those skilled in the art. Assayable amounts of a compoundof formula (I) will generally vary from about 0.001% to about 75% of thecomposition by weight. Inert carriers can be any material which does notdegrade or otherwise covalently react with a compound of formula (I).Examples of suitable inert carriers are water; aqueous buffers, such asthose which are generally useful in High Performance LiquidChromatography (HPLC) analysis; organic solvents, such as acetonitrile,ethyl acetate, hexane and the like; and pharmaceutically acceptablecarriers or excipients.

More particularly, the present invention provides pharmaceuticalcompositions comprising a therapeutically effective amount of a compoundof formula (I) in admixture or otherwise in association with one or morepharmaceutically acceptable carriers or excipients.

The pharmaceutical compositions are prepared in a manner well known inthe pharmaceutical art. The carrier or excipient may be a solid,semi-solid, or liquid material which can serve as a vehicle or mediumfor the active ingredient. Suitable carriers or excipients are wellknown in the art. The pharmaceutical composition may be adapted for oralor parenteral use and may be administered to the patient in the form oftablets, capsules, suppositories, solution, suspensions, or the like.

The compounds of the present invention may be administered orally, forexample, with an inert diluent or with an edible carrier. They may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the compounds may be incorporatedwith excipients and used in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, chewing gums and the like. Thesepreparations should contain at least 4% of the compound of theinvention, the active ingredient, but may be varied depending upon theparticular form and may conveniently be between 4% to about 70% of theweight of the unit. The amount of the compound present in compositionsis such that a suitable dosage will be obtained. Preferred compositionsand preparations according to the present invention are prepared so thatan oral dosage unit form contains between 5.0-300 milligrams of acompound of the invention.

The tablets, pills, capsules, troches and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose, disintegrating agents such as alginic acid, Primogel, cornstarch and the like; lubricants such as magnesium stearate or Sterotex;glidants such as colloidal silicon dioxide; and sweetening agents suchas sucrose or saccharin may be added or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or a fatty oil. Otherdosage unit forms may contain other various materials which modify thephysical form of the dosage unit, for example, as coatings. Thus,tablets or pills may be coated with sugar, shellac, or other entericcoating agents. A syrup may contain, in addition to the presentcompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically pure and non-toxic in theamounts used.

For the purpose of parenteral therapeutic administration, the compoundsof the present invention may be incorporated into a solution orsuspension. These preparations should contain at least 0.1% of acompound of the invention, but may be varied to be between 0.1 and about50% of the weight thereof. The amount of the inventive compound presentin such compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention are prepared so that a parenteral dosage unit contains between5.0 to 100 milligrams of the compound of the invention.

The solutions or suspensions may also include the one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

The present invention is also directed to combinations of the HIVprotease-inhibitory compounds with one or more agents useful in thetreatment of AIDS, such as, for example, with known antiviral agentssuitable for treating HIV 1 and HIV 2 viral infections, e.g., AZT, withor without a PNPase inhibitor, or in conjunctive therapy with DDI and aPNPase inhibitor.

The compounds of this invention may be assayed for their HIV-proteaseinhibition using the following techniques.

Preparation of Retroviral Enzyme and Assay for Inhibition of theProtease

A) Preparation of Retroviral Enzyme

To prepare the recombinant protease, the HIV protease is expressed viaE. Coli by the published work of C. Guenet, et al., in European Journalof Pharmacology, Molecular Pharmacology Section, 172, 443-451,(1989).The recombinant enzyme was partially purified according to Darke, P. L.et al., J. Biol. Chem., 256, 2307 (1989).

B) Enzyme Assay

The specific activity of the partially purified protease is in the rangeof 10-100 units per mg protein (one unit is defined as the amount ofenzyme that will cleave one mole of H-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH₂per minute at 37° C. under the assay conditions). HIV-1 is assayedagainst the octapeptide H-Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH₂. The reactionis performed in 0.1 mL of a buffer containing 0.05 M sodium acetate, 0.5M sodium chloride, 1 mM EDTA, 0.5% BSA, 5% ethyleneglycol, 10% glycerol,pH 5.5. The reaction is stopped after an incubation time of 1 hour at37° C. via quenching with perchloric acid (final concentration 0.4M) andcetrifuged (Eppendorf) for 5 minutes. The products of the reaction,H-Ser-Gln-Asn-Tyr-OH (P₁) and H-Pro-Ile-Val-NH₂ (P₂), are analyzed byHPLC on a C₁₈ column (Ultrasphere ODS, 4.6×150 mm, 5 mm, Beckman), byintegration of the corresponding peak areas. The elution is performedwith an acetonitrile gradient (5% acetonitrile, pH 3.0 to 60%acetonitrile, pH 3.0 in 10 minutes, at a flow rate of 1 mL/min;retention times: P₁ =6 minutes, P₂ =7 minutes and S=8.3 minutes). K_(i)values are determined from a Dixon plot (l/v versus [I]), see Segal, I.H., Enzyme Kinetics, 109 (1975). The K_(i) for[(9S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-(phenylmethyl)-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide=10to 30 nM.

By following the techniques referenced above, as well as by utilizationof other known techniques, as well as by comparison with compounds knownto be useful for treatment of the above-mentioned disease states, it isbelieved that adequate material is available to enable one of ordinaryskill in the art to practice the invention.

As with any group of structurally related compounds which possesses aparticular generic utility, certain groups and configurations arepreferred for compounds of formula (I) in their end-use application.

Compounds of formula (I) wherein X is 1 are generally preferred.Compounds of formula (I) wherein P₃ is --CH₂ CO₂ H, --CH₂ CONH₂, --CH₂(CH₂)₃ NH₂, --CH₂ CH₂ CO₂ H, --CH₂ CH₂ CONH₂, benzyl and ##STR28## aregenerally preferred. Compounds of formula (I) wherein P₂ is --CH(CH₃)₂,cyclopentyl and phenyl are generally preferred. Compounds of formula (I)wherein the configuration about the carbon atom in the cyclic structureto which P₃ is attached is in the D configuration are generallypreferred. Compounds of formula (I) in which R₁ is hydrogen and R₂ isbenzyl, 2-pyridyl, 3-pyridyl and ##STR29## are generally preferred.

Examples of compounds according to the present invention are thefollowing:

1)[(9S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-(phenylmethyl)-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide;

2)[(9S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-[2-methyl-1-[(phenylmethoxy)methyl]propyl]-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide;

3)N-Benzyl-3-(6-benzyl-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl)-2,2-difluoro-3-oxo-propionamide;

4)3-[12-(Benzylcarbamoyl-difluoro-acetyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-6-yl]-propionicacid;

5)3-[6-(4-Amino-butyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triaza-bicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl]-N-benzyl-2,2-difluoro-3-oxo-propionamide;

6)N-Benzyl-3-[6-(2-carbamoylethyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl]-2,2-difluoro-3-oxo-propionamide;

7)[12-(Benzylcarbamoyl-difluoro-acetyl)-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-6-yl]-aceticacid;

8)N-Benzyl-3-(6-carbamoylmethyl-9-isopropyl-4,7,10-trioxo-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl)-2,2-difluoro-3-oxo-propionamide;

9)N-Benzyl-2,2-difluoro-3-(9-isopropyl-4,7,10-trioxo-6-pyridin-3-ylmethyl-2-oxa-5,8,11-triazabicyclo[12.2.2]octadeca-1(17),14(18),15-trien-12-yl)-3-oxo-propionamide;

and the stereoisomers, hydrates and pharmaceutically acceptable saltsthereof.

What is claimed is:
 1. A compound of the formula: ##STR30## and thestereoisomers, hydrates, and pharmaceutically acceptable salts thereofwhereinP₂ is C₁₋₆ alkyl, cyclopentyl, hydroxy C₁₋₆ alkyl, phenyl, benzylor 3-tetrahydrofuryl; P₃ is selected from the group consisting ofhydrogen, --CH₃, --CH(CH₃)₂, --CH₂ CH(CH₃)₂, --CH(CH₃)(CH₂ CH₃), --CH₂SH, --CH₂ CH₂ SCH₃, --CH₂ OH, --CH(CH₃)OH, --CH₂ (CH₂)₃ NH₂, --CH₂(CH₂)₂ NHC(═NH)NH₂, --CH₂ CO₂ H, --CH₂ CH₂ CO₂ H, --CH₂ CONH₂, --CH₂ CH₂CONH₂, benzyl, ##STR31## R₁ is hydrogen, C₁₋₁₅ alkyl, hydroxy C₁₋₁₅alkyl, CH([(CH₂)_(d) -O--CH₂ ]_(f) -R₇)₂, CH₂ Si(CH₃)₂ (R₈), PDL,--(C₁₋₆ alkylene)--OR₄, CH(Y)(Z), ##STR32## wherein PDL is --(CH₂)_(a)-2-, 3- or 4-pyridyl; Y is hydroxy C₁₋₁₅ alkyl, C₁₋₆ alkyl or (CH₂)_(e)-C₆ H₄ -(V)_(e') ; Z is (CH₂)_(d) -O--CHO, C₁₋₆ alkylene-O-(CH₂)_(d)-(O--CH₂ --CH₂)_(e) -O--C₁₋₆ alkyl, CHO, CO₂ R₄, CONHR₄, (CH₂)_(d)-O--(CH₂)_(d') -R₅, (CH₂)_(e) -OR₄ or ##STR33## wherein V is OR₄ orhydroxy C₁₋₆ alkylene; provided that d'=2 when R₅ is piperazinyl,substituted piperazinyl, piperidyl or morpholinyl; R₂ is as defined forR₁ with the proviso that R₂ is other than hydrogen when R₁ is hydrogen,or R₁ and R₂ taken together with the nitrogen atom to which they areattached are selected from the group consisting of; ##STR34## R₄ ishydrogen, C₁₋₆ alkyl, phenyl or benzyl; R₅ is piperazinyl, substitutedpiperazinyl, piperidyl, morpholinyl, pyridyl, pyrazinyl, pyrimidinyl orphenyl, wherein substituted piperazinyl is piperazinyl substituted onone nitrogen atom thereof with CHO, C(O)NHR₄, C₁₋₄ alkyl or CO₂ R₄ ; R₇is pyrimidyl, pyridyl, pyrazinyl or phenyl; R₈ is C₁₋₆ alkylene, hydroxyC₁₋₆ alkyl or C₁₋₆ alkyl; a is zero, 1, 2 or 3; b is zero or 1; d and d'are each independently 1 or 2; e and e' are each independently zero, 1or 2; f is zero or one; and x is 1, 2, 3, or
 4. 2. A compound accordingto claim 1 wherein X is
 1. 3. A compound according to claim 2 wherein P₂is --CH(CH₃)₂.
 4. A compound according to claim 3 wherein R₁ is hydrogenand R₂ is benzyl.
 5. A compound according to claim 3 wherein R₁ ishydrogen and R₂ is 2-(3-methyl-1-phenylmethoxy)butyl.
 6. A compoundaccording to claim 4 wherein P₃ is hydrogen.
 7. A compound according toclaim 4 wherein P₃ is CH₂ CO₂ H.
 8. A compound according to claim 4wherein P₃ is CH₂ CONH₂.
 9. A compound according to claim 4 wherein P₃is CH₂ CH₂ CO₂ H.
 10. A compound according to claim 4 wherein P₃ is CH₂CH₂ CONH₂.
 11. A compound according to claim 4 wherein P₃ is benzyl. 12.A compound according to claim 4 wherein P₃ is ##STR35##
 13. A compoundaccording to claim 4 wherein P₃ is --CH₂ (CH₂)₃ NH₂.
 14. A compoundaccording to claim 4 wherein the carbon atom in the macrocyclic ring towhich P₃ is attached, is in the D configuration.
 15. A compoundaccording to claim 1 wherein the compound is[9(S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-(phenylmethyl)-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide.16. A compound according to claim 1 wherein the compound is[9(S),12(S)]-α,α-Difluoro-9-(1-methylethyl)-β,4,7,10-tetraoxo-N-[2-methyl-1-[(phenylmethoxy)-methyl]propyl]-2-oxa-5,8,11,-triazabicyclo[12.2.2]octadeca-14,16,17-triene-12-propanamide.